This proposal is aimed toward a long term goal of understanding the mechanism of germ line establishment, by focusing on Vasa (Vas), a maternally expressed protein essential for germ cell specification in Drosophila that has been implicated in translational regulation of specific mRNAs through an interaction with elF5B. Vas is essential for mammalian spermatogenesis, making this work highly relevant for understanding the etiology of male infertility. The human Vas homolog has also been developed as an early diagnostic marker for several germ cell tumors including seminomas and dysgerminomas. The main challenges now are to understand how Vas interacts with proteins involved in general cellular processes to accomplish its functions in development. 5 specific aims are proposed for the upcoming term of the grant. The first involves identification of Vas-associated RNAs using cross-linking and immunoprecipitation. mRNAs that are recovered will be assessed for whether they accumulate in the pole plasm or pole cells, mutations in the genes encoding them will be assessed for function in germ cell specification, and whether their translation is sensitive to Vas and the Vas-elF5B interaction will be determined. The second aim involves an investigation of the function of Vas in translation, using in vivo and in vitro approaches. The third aim concerns a further characterization of the interactions between Vas, Osk, and several proteins identified in coimmunoprecipitation experiments. The next aim focuses on Gus, a Vas-interacting protein implicated in its localization to the pole plasm. Targeted mutations in Gus that abrogate Vas interaction and that disrupt conserved domains will be produced, and their phenotypes examined. Possible links between Gus and the cytoskeleton will also be investigated. Finally, in the 5th aim the relationship between Tud, a protein essential downstream of Vas for germ line establishment, Cup, a translational regulator, TACC, a microtubule binding protein, and Dynein heavy chain will be examined.